Project Abstract Electrospray Ionization Mass Spectrometry (ESI-MS) is a biochemical analysis workhorse, with Life Sciences equipment sales: $3.5 billion annually1 (see Bibliography). It is well developed for the analysis of proteins, peptides, DNA and macro-molecules, with sizes below 15 nm, but is not yet able to cover the next register in sizes, the virus-range (20-250 nm; Scheme 1). Mobility-based size analysis of charge-reduced electrosprayed particles has offered an alternative window to the viral size range since 1996. However, in spite of outstanding demonstrated sensitivity (single particle count) and quantification ability (far superior to other first line non- biological tools such as electron microscopy), the limited resolving power of its commercial form (TSI Inc.) has thwarted its widespread adoption. This SBIR promises to fulfill critical unmet need for quick, general-purpose, virus particle size-analysis, with greatly improved resolving power, resulting in sharp distinction of Nanoscopic ?Virion? (and large protein complex) particles in the range 10-60 nm. High size resolution not only better distinguishes viral analytes for species identification, but also makes viral peaks emerge distinctly from the background, especially because in insect vector Superinfections one dominant viral contagion population suppresses all others through viral interference2. We anticipate a size resolving power in excess of 30-50, comparable with the outstanding recent developments in virus MS by Jarrold, 3 though in a far simpler, economical, faster and more sensitive device. NanoEngineering Corporation?s (NEC) current portable precision High Resolution (Ion) Mobility Analysis of ESI-aerosolized virus particles (NanoRanger? 60) instrument offers already a resolution >30 over insect virus range 20-60 nm, with potential extension to 20-120 nm to span nearly all respiratory viruses. This resolution well exceeds limits of conventional laser-particle scattering (7)4.We propose three main developments: (i) Exploit systematically the existing high resolution Differential Mobility Analyzer (DMA) in collaboration between NEC, Yale and BVS Inc. to develop improved protocols for sample preparation, especially of insect viri. This collaboration should be most fruitful, as no high resolution mobility instrument has been previously available to guide improved sample preparation. (ii) Further develop this DMA to increase its resolving power. The improved DMA will be tested at Yale with Bio-Safety Level 1 viral particles having a fixed size (i.e. a capsid). (iii) A final vital proposed development is to drastically advance the Tech Readiness Level of the existing size-analysis DMA instrumentation, such that it can reliably be used by virologists during Phase II of this program. Results will qualify this SBIR tech for Phase II/IIB trials for field-surveillance of mosquitos-borne contagions (Zika, Dengue). Phase II will be accomplished via installation of a beta-unit at Scripps Research Institute?s BLS 2+ facility (FL), a stepping stone to deployment with early-adopters at three mosquito labs: 1) U. of S. FL; 2) USDA; 3) FL Medical Entomology Lab and beyond. DMA Phase II extension will span to 120 nm. Phase III will target commercial manufacturing and marketing of patented NanoRanger? ESI-DMA instruments. ESI-Mass Spectrometry ? 15 nm diameter Macromolecules 0 15 Underserved Size-Gap 15 ? 300 nm Virus Size Range 20 to ~ 250 nm Optical Microscopes ? 300 nm diameter Single Cell Organisms etc. 300 Particle Size (nm) Scheme 1. Unmet market need for quick, low-cost virion-nanoparticle size measurement may be fulfilled by ESI-DMA. SBIR success could serve as springboard to general adoption of this instrument in life sciences and biomedicine.